(1) Field of the Invention
This invention relates generally to the prevention of coagulation. More particularly, this invention relates to compositions and methods for preventing coagulation by inhibiting binding of factor IXa to factor VIIIa, and applications utilizing these compositions and methods, including treating patients in need of anti-coagulants, preventing coagulation in blood samples, and detecting factor VIIIa.
(2) Description of the Related Art
Two common causes of abnormal bleeding are deficiencies of factor VIII (hemophilia A) or factor IX (hemophilia B). Factor IX, a vitamin K-dependent protein, is synthesized by hepatocytes as a precursor molecule of 461 residues containing a 28 residue signal propeptide and an 18 residue leader propeptide (Yoshitake et al., 1985, Biochemistry 24, 3736-3750). During biosynthesis, the nascent protein undergoes several posttranslational modifications, resulting in a single-chain protein consisting of 415 amino acids and containing 17% carbohydrate by weight (DiScipio et al., 1978, J. Clin. Invest. 61, 1528-1538). The mature protein circulates in blood as a zymogen of Mr 57,000.
Factor IX is activated during physiologic clotting to the two-chain, disulfide-linked serine protease, factor IXa, by VIIa/Ca2+/tissue factor (TF) or by factor XIa/Ca2 + (Davie et al., 1991, Biochemistry 29, 10363-10370). The domain organization of factor IXa is similar to those of the other two enzymes (factors VIIa and Xa) involved in the TF-induced coagulation and to that of an anticoagulant enzyme termed activated protein C. The light chain of IXa consists of an amino-terminal xcex3-carboxyglutamic acid domain (xe2x80x9cGla domainxe2x80x9d, residues 1-40 out of which 12 are xcex3-carboxyglutamic acid residues), a short hydrophobic segment (residues 41-46), and two epidermal growth factor (EGF)-like domains (EGF1 residues 47-85, and EGF2 residues 86-127) whereas the heavy chain contains the carboxy-terminal serine protease domain with trypsin-like specificity (Id.; Brandstetter et al., 1995, Proc. Natl. Acad. Sci. USA 92, 9796-9800). Activation peptide (AP) of residues 145-180 which is released upon conversion of factor IX to IXa is rich in carbohydrate and is the least conserved region in IX from different species (Sarkar et al., 1990, Genomics 6, 133-134). Factor IXa hence formed converts factor X to Xa in the coagulation cascade; for a biologically significant rate, this reaction requires Ca2+, phospholipid and factor VIIIa.
Based upon the crystal structure of the Gla domain of factor VIIa (Banner et al., 1996, Nature 380, 41-46) and the Ca2+-binding properties of factor X (Sabharwal et al., 1997, J. Biol. Chem. 272, 22037-22045.), it would appear that this domain in IXa possesses several low to intermediate affinity Ca2+-binding sites. In addition, the EGF1 and the protease domain each possess one high affinity Ca2+-binding site (Rao et al., 1995, Cell 82, 131-141; Bajaj et al., 1992, Proc. Natl. Acad. Sci. USA 89, 152-156). The Ca2+-loaded conformer of the Gla domain binds to phospholipid vesicles (Freedman et al., 1996, J. Biol. Chem. 271, 16227-16236) and the EGF1 domain of IX is required for its activation by VIIa/Ca2+/TF (Zhong et al., 1994, Proc. Natl. Acad. Sci. USA 91, 3574-3578). Further, Ca2+-binding to the EGF1 domain has been reported to promote enzyme activity and factor VIIIa binding (Lenting et al., 1996, J. Biol. Chem. 271, 25332-25337). The role of the EGF2 domain is not clear but may be involved in binding to platelets and in factor X activation (Ahhmed et al., 1995, Biochem. J. 310, 427-431). Finally, the protease domain is thought to play a primary role in binding to factor VIIIa (Astermark et al., 1994, J. Biol. Chem. 269, 3682-3689; O""Brien et al., 1995, J. Biol. Chem. 270, 27087-27092; Bajaj et al., 1993, Methods Enzymol. 222, 96-128).
Recently, it has been demonstrated that mutations in the protease domain Ca2+-binding ligands decrease the affinity of factor IXa for factor VIIIa by xcx9c15-fold and that proteolysis at R318-S319 [residues 150-151 in the chymotrypsin numbering system] in the autolysis loop results in a further decrease in this interaction by xcx9c8-fold (Mathur et al., 1997, J. Biol. Chem. 272, 23418-23426). Since residues in the protease domain Ca2+-binding loop as well as those in the autolysis loop may not directly participate in binding to factor VIIIa (Hamaguchi et al., 1994, Blood 84, 1837-1842), it can be hypothesized that Ca2+ binding to the protease domain and integrity of the autolysis loop stabilize yet another region in this domain of factor IXa that directly interacts with factor VIIIa.
The identification of the factor VIIIa binding site on factor Ixa would be useful for identifying agents with anti-coagulation activity, as well as for designing treatments which prevent excessive thrombosis in a patient, and preventing coagulation of blood samples.
In accordance with the present invention, the inventor herein has succeeded in identifying a crucial region in factor IXa which binds to factor VIIIa. Compositions which comprise critical amino acids of this region, but which do not activate factor X, bind to factor VIIIa, preventing the binding of factor IXa, thus preventing coagulation. These compositions can thus be used to prevent thrombosis in patients in need thereof. The compositions can also be used to prevent coagulation in a blood sample, and to detect and quantify factor VIIIa in a sample.
Thus, one embodiment of the present invention is directed to a polypeptide, or derivative thereof, including peptidomimetics and nonpeptidomimetics, comprising DRXaaT or derivatives, where Xaa is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity. This polypeptide or derivative comprises the three amino acids essential to the factor VIIIa binding site in factor IXa. Preferably, this polypeptide, or derivative binds to factor VIIIa but does not activate factor X. The polypeptide or derivative also preferably comprises from 4 to 20 contiguous amino acids. Most preferred polypeptides or derivatives comprise SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR), which are sequences in the factor VIIIa binding site in factor IXa.
Another embodiment of the present invention is directed to an isolated and substantially purified polypeptide or derivative thereof, comprising DRXaaT where Xaa is any amino acid other than alanine. Preferred polypeptides or derivatives here have from 4 to 20 amino acids. The polypeptide or derivative also preferably binds to factor VIIIa but does not activate factor X.
In an additional embodiment, the present invention is directed to an isolated and purified nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide comprising DRXaaT, where Xaa is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity, or the complement thereof. Preferably, the polypeptide comprises SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR).
Additionally, the present invention is directed to a method for identifying an agent having anti-coagulation activity. The method comprises determining whether a candidate agent displaces the binding of a polypeptide comprising SEQ ID NO:2 (DRAT) from factor VIIIa, wherein the polypeptide preferably has from 4 to 20 amino acids, but may be longer, up to and including the entire factor IXa molecule. Preferably, the polypeptide is a labeled polypeptide and the determining step comprises detecting the labeled polypeptide displaced from factor VIIIa. Preferred labels comprise a radioactive or a fluorescent moiety.
In an additional embodiment, the present invention is directed to a method of treatment to prevent thrombosis in a patient in need thereof. The method comprises administering to the patient a polypeptide or derivative thereof comprising DRXaaT, where Xaa, is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity. The preferred polypeptide or derivative binds to factor VIIIa but does not activate factor X. More preferably, the polypeptide has from 4 to 20 amino acids. Most preferred polypeptides comprise SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR). An antibody that specifically binds to these sequences would also be expected to prevent thrombosis. Such an antibody could also be utilized in place of the above polypeptides or derivatives in the embodiments described below.
In still another embodiment, the present invention is directed to a method of treatment to prevent thrombosis in a patient in need thereof. The method comprises selecting an agent having anti-coagulation activity, wherein the selecting comprises testing the agent for activity in displacing the binding of Factor IXa from Factor VIIIa without activating Factor X, and administering the agent to the patient.
An additional embodiment of the present invention is a method of preventing coagulation in a blood sample. The method comprises adding to the sample, in sufficient quantity to prevent coagulation, a polypeptide or derivative thereof comprising DRXaaT, where Xaa is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity. A preferred polypeptide or derivative is capable of binding to factor VIIIa but does not activate factor X. More preferably, the polypeptide or derivative has from 4 to 20 amino acids or derivatives. Most preferable polypeptides or derivatives comprise SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR).
Additionally, the present invention is directed to a method of detecting factor VIIIa in a sample. The method comprises (a) contacting the sample with a polypeptide or derivative thereof comprising a covalently attached detectable moiety and DRXaaT, where Xaa is any amino acid, and wherein the polypeptide or derivative has anti-coagulation activity, and (b) determining whether the polypeptide or derivative is binding factor VIIIa from the sample. Preferably, the detectable moiety is radioactive or fluorescent.